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Ultra fine super self-nanoemulsifying drug delivery system (SNEDDS) enhanced solubility and dissolution of indomethacin
Journal of Molecular Liquids 180 (2013) 89–94
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Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq
Ultra fine super self-nanoemulsifying drug delivery system (SNEDDS) enhanced solubility and dissolution of indomethacin Faiyaz Shakeel a, b,⁎, Nazrul Haq a, b, Mahmoud El-Badry b, Fars K. Alanazi b, c, Ibrahim A. Alsarra a, b a b c
Center of Excellence in Biotechnology Research, King Saud University, Riyadh, Saudi Arabia Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia Kayyali Chair for Pharmaceutical Technology, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
a r t i c l e
i n f o
Article history: Received 10 October 2012 Received in revised form 4 January 2013 Accepted 16 January 2013 Available online 30 January 2013 Keywords: Self-nanoemulsifying drug delivery system Indomethacin Dissolution Solubility enhancement
a b s t r a c t The aim of the present investigation was to develop and evaluate ultra fine super self-nanoemulsifying drug delivery system (SNEDDS) of indomethacin (IND) to enhance its solubility as well as in vitro dissolution rate. Different SNEDDS formulations of IND were prepared by aqueous phase titration method. Prepared SNEDDS were subjected to different thermodynamic stability tests. Thermodynamically stable SNEDDS was selected for self-nanoemulsification efficiency test. Selected formulations were characterized in terms of droplet size distribution, viscosity and refractive index. Finally, selected SNEDDS (F1–F9) were subjected to in vitro dissolution/drug release studies. Droplet size and viscosity of formulation F1 was found to be lowest as compared to other formulations. The results of zeta potential indicated the formation of stable SNEDDS. In vitro drug release studies showed 98.4% release of IND from optimized formulation F1. The initial drug release profile of IND from optimized formulation F1 was found to be much faster than marketed IND capsule. The results of solubility studies showed around 4573 fold enhancement in solubility in F1 formulation compared to its aqueous solubility. These results indicated that developed SNEDDS could be successfully used for self-nanoemulsifying drug delivery of IND in order to enhance its solubility as well as in vitro dissolution rate. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Indomethacin (IND) is a potent lipophilic nonsteroidal antiinflammatory drug (NSAID) which has been recommended for the treatment of various kinds of pain associated with arthritis, gout and collagen diseases [1–3]. Currently many therapies like oral, topical and parenteral/injectional are used clinically for the treatment of these diseases. Like other NSAID, IND produces gastric/peptic ulcers upon frequent/chronic administration [4,5]. As per the biopharmaceutical classification (BCS) system, IND possesses poor solubility and high permeability (BCS class II drug). Poor solubility of drugs could result in poor in vivo absorption and weak bioavailability, therefore enhancement in solubility and dissolution rate is important in formulation development. Ultra fine super self-nanoemulsifying drug delivery systems (SNEDDS) are clear, transparent, isotropic system of drug, oil, surfactant and cosurfactant, which forms ultra fine nanoemulsions (usually less than 50 nm in size) upon mild agitation, followed by dilution with an aqueous media or gastrointestinal
⁎ Corresponding author at: Center of Excellence in Biotechnology Research, King Saud University, Riyadh, Saudi Arabia (KSA). Tel.: +966 537507318. E-mail addresses: [email protected], [email protected] (F. Shakeel). 0167-7322/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molliq.2013.01.008
(GI) fluids [6–8]. In the last decade, many molecular liquid based systems like microemulsions, nanoemulsions and SNEDDS etc. have been investigated as novel nanovehicles for many hydrophobic drugs to enhance their thermodynamic stability, solubilization capacity, in vitro dissolution, drug delivery potential, therapeutic efficacy as well as in vivo bioavailability [1–3,6–12]. Some formulation approaches like emulsions, nanoemulsions, nanoparticles and SNEDDS have been investigated for nasal, trasnsdermal, ocular and oral drug delivery of IND in order to enhance its permeation, solubilization and dissolution [1–3,13–15]. Ultra fine super SNEDDS have not been investigated as nanovehicles for oral delivery of IND. When ultra fine super SNEDDS administered orally, they spread rapidly in an aqueous media (GI fluids) providing the hydrophobic drugs in nanometer size range, which in turn enhance the drug dissolution and bioavailability by increasing their aqueous solubility. Therefore, the aim of present study was to develop, characterize and evaluate ultra fine super SNEDDS for oral delivery of IND in order to enhance its solubility as well as dissolution rate which in turn enhance the bioavailability and reduce adverse effects associated with conventional oral therapy of IND. The significant advantage of the present study is that ultra fine super SNEDDS were prepared by spontaneous emulsification (low energy emulsification) method using those materials which are pharmaceutically acceptable and falls under generally regarded as safe category.
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2. Experimental
required amount of Smix was added with drop-wise water addition till an apparent and clear solution was obtained.
2.1. Materials 2.6. Characterization and optimization of SNEDDS IND was obtained as a gift sample from Alfa Aesar, A Johnson Metthey Company (Ward Hill, MA). Labrafil, Lauroglycol-90, Caproyl-90, Labrasol and Transcutol-HP were kindly gifted by Gattefossé (France). HPLC grade ethyl acetate (EA) and Tween-80 were purchased from BDH chemicals (UK). All these materials were of high purity (>99%). All other chemicals used in the study were of analytical reagent (AR) grade. 2.2. Analytical methodology IND in solubility study samples as well as in dissolution samples was analyzed by in-house developed green reversed phase high performance liquid chromatography (RP-HPLC) method in which an environmentally benign green solvent EA (100%) was used as a mobile phase. Green chromatographic identification of IND was performed on Waters HPLC system (Waters, USA) using Lichrosphere 250 × 4.6 mm reverse phase (RP) C8 column (Phenomenex, USA) having a 5 μm packing as a stationary phase. The elution was performed at a flow rate of 1.0 ml/min at 318 nm. Samples (10 μl) were injected using a Waters auto sampler (unpublished data). 2.3. Screening of components for ultra fine super SNEDDS preparation Pharmaceutically acceptable and generally regarded as safe category components were selected for the development of ultra fine super SNEDDS of IND. Selected oils, surfactants and cosurfactants were screened based on solubility of IND in these components. As per our previous report, highest solubility of IND was obtained in Labrafil (as oil phase), Tween-80 (as surfactant) and Transcutol-HP (as cosurfactant) [2]. Therefore, Labrafil, Tween-80, Transcutol-HP and distilled water were selected as oil phase, surfactant, cosurfactant and aqueous phase respectively for the development of optimal ultra fine super SNEDDS of IND. 2.4. Nanophasic map construction and optimization of ultra fine super SNEDDS In order to find out the concentration range of various components for the existence zones of SNEDDS, pseudo-ternary phase diagrams were constructed using aqueous phase titration method [16–18]. Surfactant and co-surfactant (Smix) were premixed in different weight ratios (1:0, 1:1, 1:2, 1:3, 2:1, and 3:1). The concentration of each Smix was 25 g. For each phase diagram, oil phase and specific Smix ratio were mixed thoroughly in different weight ratios ranging from 1:9 to 9:1. Slow titration with aqueous phase was done for each combination of oil and Smix separately [17]. The physical state of prepared SNEDDS was marked on a pseudo-ternary component based phase diagram with one axis representing the aqueous phase, oil phase and the third representing a mixture of surfactant and cosurfactant at fixed weight ratios (Smix ratio). 2.5. Formulation development IND encapsulated nanoemulsions were prepared as similar to our previous research [2]. From the pseudo-ternary phase diagram, nanophasic regions were pointed out and different formulations from this location were chosen for the present study. Considering phase diagrams, maximum nanophasic regions were exposed by 2:1 ratio of Tween-80 and Transcutol-HP in varying proportions. Almost an entire range of nanoemulsions (NE) occurrence in the phase diagram was taken into account and varied oil compositions with minimum surfactant concentration were selected. 25 mg of IND was taken and
2.6.1. Thermodynamic stability test In search of robust formulation and to overcome the problem of metastable formulation, thermodynamic stability tests were performed [16]. Screened formulations were subjected to different thermodynamic stability tests viz. centrifugation, heating & cooling cycle and freeze– pump–thaw cycles as reported in our previous article [16–18]. 2.6.2. Droplet size and zeta potential measurement Mean droplet size and polydispersity index (PI) of screened SNEDDS (F1–F9) were determined using Brookhaven 90 plus particle size analyzer (Brookhaven Instrument Corporation, NY) which was based on light scattering at room temperature (25 °C) at a scattering angle of 90°. The samples of IND-loaded SNEDDS were suitably diluted with distilled water (1:200), sonicated and filtered through 0.45-μm membrane filter. 3 ml of each SNEDDS was taken in an acrylic square. The refractive index (RI) and viscosity of the medium were set at 1.33 and 0.89 cp, respectively. Zeta potential of screened SNEDDS (F1-F9) was determined using Brookhaven 90 plus zeta potential analyzer (Brookhaven Instrument Corporation, NY) using glass cells. 2.6.3. Viscosity and refractive index measurement Viscosity of IND loaded SNEDDS was measured by Brookfield viscometer at 25 ± 1 °C. Refractive index of SNEDDS was determined without dilution using Abbes type refractometer at 25 ± 1 °C. 2.7. Self-nanoemulsification efficiency test Thermodynamically stable SNEDDS were further subjected to self-nanoemulsification efficiency test. The self-nanoemulsification efficiency of IND loaded SNEDDS was assessed using a standard USP XXII dissolution apparatus 2 as reported previously [16]. The self-nanoemulsification performance of each SNEDDS was visually assessed using different grading systems like grades A, B, C, D and E as reported previously [16,19,20]. Formulations those passed self-nanoemulsification efficiency test in grades A and B were selected for further evaluation. 2.8. In vitro dissolution/drug release studies In vitro dissolution/drug release studies were performed in 900 ml of distilled water using USP XXIV method (Dissolution apparatus # 2, at 50 rpm and 37± 0.5 °C). One ml of each SNEDDS (containing 25 mg of IND) was filled in transparent hard gelatin capsules. Samples (3 ml) were withdrawn at regular time intervals (0, 2, 5, 10, 15, 20, 30, 45 and 60 min) and aliquot amount of distilled water was replaced every time. The release of IND from SNEDDS formulation was compared with the marketed capsule formulation. The samples were analyzed for the drug content using in-house developed green RP-HPLC method at the wavelength of 318 nm. 2.9. Determination of IND solubility in distilled water and optimized SNEDDS F1 The saturated solubility of IND in distilled water and optimized SNEDDS F1 was determined by in house developed RP-HPLC method at 318 nm using pure EA as mobile phase (unpublished data). IND in excess amount was added in distilled water and formulation F1 in glass vials (capacity 5 ml) in triplicate. These glass vials were kept in a mechanical Grant shaking water bath OLS 200 (Grant Scientific, UK) at 37 ± 1 °C for 3 days to get equilibrium. After 3 days, samples were filtered by 0.45-μm membrane filter and diluted suitably with
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distilled water. Filtered and diluted samples were injected in HPLC instrument for the quantification of IND by RP-HPLC method at 318 nm. 2.10. Statistical analysis In vitro drug release data of SNEDDS formulations (F1–F9) & marketed capsule and characterization parameters were statistically evaluated by one way analysis of variance (ANOVA) using Dunnett's test to evaluate the significant differences. The statistical software SPSS Version 11 was used to analyze statistical data. 3. Results and discussion 3.1. Screening of components Based on solubility profile of IND in oil, surfactants and cosurfactant; Labrafil, Tween-80 and Transcutol-HP were selected as oil phase, surfactant and cosurfactant respectively for the development of optimal ultra fine super SNEDDS of IND. Distilled water was selected as aqueous phase. The saturation solubilities of IND in Labrafil, Tween-80 and Transcutol-HP were found to be 35.42± 1.12, 72.23 ± 2.91 and 188.97± 3.29 mg/ml, respectively [2]. 3.2. Nanophasic diagram construction and optimization of ultra fine super SNEDDS Pseudo-ternary phase diagrams for ultra fine super SNEDDS were constructed separately for each Smix ratio for the optimization of formulations (Fig. 1). From the phase diagram, it was clearly observed that Smix 1:0 (Fig. 1a) had very low SNEDDS zones. The maximum concentration of oil that was solubilized by this ratio (1:0) was found to be 9% w/w by utilizing very high percentage of Smix (82% w/w). But in the case of Smix 1:1 (Fig. 1b), when the cosurfactant (Transcutol-HP)
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was used along with the surfactant (Tween-80), the SNEDDS zones were found to be increased significantly as compared to 1:0. The maximum concentration of oil that was solubilized by this ratio in the phase diagram was found to be 22% w/w by incorporating Smix around 52% w/w, itself showed the domination of cosurfactant (Transcutol-HP) along with surfactant in nanosizing. The increased SNEDDS zones in 1:1 ratio could be due to the synergistic effects of surfactant and cosurfactant. When the Smix ratio of 2:1 (Fig. 1c) was studied, the SNEDDS zones were increased further as compared to 1:1 ratio. The maximum concentration of oil that was solubilized by 2:1 ratio was found to be 28% w/w by incorporation of Smix around 41% w/w. When the concentration of the surfactant was increased further with respect to the cosurfactant (Smix ratio 3:1, Fig. 1d), the SNEDDS zones were found to be decreased as compared to 2:1 as well as 1:1 ratio. Possibly, this could be a liquid crystalline phase generated by Tween-80 (surfactant) and it was not pacified by the present amount of Tarnscutol-HP (cosurfactant). This liquid crystalline phase could be due to the significant enhancement in the specific conductivity and viscosity of the systems in the phase diagrams [21,22]. There is no need to increase the concentration of the surfactant with respect to the cosurfactant further because the SNEDDS zone started decreasing now (no need to study the Smix ratio of 4:1 or 5:1). When the concentration of the cosurfactant was increased with respect to the surfactant (Smix ratio of 1:2 and 1:3), no SNEDDS zones were observed in the phase diagrams (data not shown). 3.3. Formulation development With regard to nanophasic maps, good SNEDDS zones were exposed by Smix ratio of 1:1, 2:1 and 3:1. Maximum SNEDDS zones were exposed by the Smix ratio of 2:1. SNEDDS formulations with different formulae from these phase diagrams (1:1, 2:1 and 3:1) were precisely selected on the evidence based performance of thermodynamic stability studies.
Fig. 1. Pseudo-ternary phase diagrams showing SNEDDS area for oil phase (Labrafil), surfactant (Tween-80), cosurfactant (Transcutol-HP) at different Smix ratios [Fig. 1a (1:0), b (1:1), c (2:1), d (3:1)] (Arrows showing increasing concentration of particular component).
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Almost the entire range of SNEDDS occurrence in the phase diagrams was covered and different oil compositions with fixed surfactant concentration (40% w/w) were precisely selected. 25 mg of IND was solubilized in required amount of Smix and oil was added with vortexing, finally distilled water was added in drop-wise manner till a clear and transparent dispersion was obtained. The prepared SNEDDS were tightly sealed in glass vials and stored at ambient temperature.
Table 2 Characterization of screened SNEDDS in terms of droplet size, polydispersity, zeta potential, refractive index and viscosity.
3.4. Characterization and optimization of SNEDDS For the development and optimization of robust SNEDDS formulations, the selected SNEDDS were subjected to different thermodynamic tests. These tests were performed qualitatively to remove metastable and thermokinetically stable SNEDDS. Those formulations, which were found to be stable at these tests, were taken for further optimization (Table 1). Table 2 summarized mean droplet size of selected SNEDDS (F1–F9) ranging from 8.7 to 23.8 nm. These results showed the ultra fine droplet size of all formulations (less than 50 nm) which could have great potential for ultra fine super SNEDDS of IND. The largest droplet size appeared in F9 which could be due to the presence of higher concentration of oil (20% w/w) and higher Smix ratio (3:1) (Table 2). When the concentration of Smix was kept constant at 40% w/w and the oil concentration was increased from 10 to 20% w/w, the droplet size was found to be increased. The droplet size of formulation F1 was found to be lowest (8.7 nm) which could be due to the lowest concentration of oil and lower Smix ratio (1:1). Droplet size of all formulations was found to be increased by increasing the Smix ratio. PI value was found to be b0.6 for all SNEDDS, indicating narrow size distribution. Formulation F1 showed least PI value (0.005) suggesting good uniformity in droplet size distribution. Highest PI value was observed with formulation F9 (0.586). Zeta potential (ZP) values of SNEDDS F1–F9 were found in the range of −30.81 to − 27.87 mV. Formulation F1 had a ZP value of − 28.96 mV (Table 2). There were no significant changes observed in ZP values of all SNEDDS [F1–F9] (p ≥ 0.05). It has been reported previously that ZP values in the range of 25–30 mV in either charge characterizes a stable formulation [23,24]. Therefore, results of ZP measurement indicated stabile formation of all SNEDDS (F1–F9) formulations. The negative net charge on ZP for all SNEDDS was possible due to the presence of fatty acids and esters in the Labrafil which was used as the oil phase for development of SNEDDS. The refractive indices (RIs) of SNEDDS (F1–F9) were found in the range of 1.334–1.339 at 25 °C (Table 2). These values of RI were o/w nature of IND loaded SNEDDS. The RI of formulation F1 was found to be 1.334. The viscosity of optimized SNEDDS (F1–F9) ranged from 24.52 to 58.16 cp. The viscosity results could be correlated with the different contribution of Labrafil, Tween-80 and Transcutol-HP utilized in stabilization of optimized formulation compositions. Viscosity of the formulation F1 was found to be lowest (24.52 cp) as compared to other formulations. The viscosity results could also be correlated with shape and geometry
Table 1 Composition of screened SNEDDS that passed thermodynamic stability and selfnanoemulsification efficiency test in grades A & B. Matrices
F1 F2 F3 F4 F5 F6 F7 F8 F9
Formulation composition (% w/w) Oil phase⁎
Smix phase
Water phase
10 15 20 10 15 20 10 15 20
40 40 40 40 40 40 40 40 40
50 45 40 50 45 40 50 45 40
⁎ Oil phase containing 25 mg of indomethacin.
Smix combination
1:1 1:1 1:1 2:1 2:1 2:1 3:1 3:1 3:1
†
Formulation
Δdm#±SD (nm)
p⁎¡
ZP (mV)
RI† ± SD
η ± SD (cps)
F1 F2 F3 F4 F5 F6 F7 F8 F9
8.70 ± 1.41 14.10 ± 1.73 17.40 ± 2.79 14.0 ± 2.15 15.40 ± 2.21 20.90 ± 3.22 15.70 ± 3.11 21.10 ± 3.54 23.80 ± 3.51
0.005 0.357 0.005 0.095 0.201 0.005 0.312 0.566 0.586
−28.96 −27.87 −29.67 −28.15 −30.21 −30.81 −29.80 −30.44 −29.55
1.334 ± 0.004 1.335 ± 0.005 1.336 ± 0.006 1.334 ± 0.005 1.336 ± 0.007 1.337 ± 0.008 1.338 ± 0.008 1.339 ± 0.009 1.339 ± 0.009
24.52 ± 3.41 34.54 ± 4.92 46.33 ± 5.17 27.56 ± 4.14 42.51 ± 5.62 51.61 ± 6.21 29.78 ± 4.58 47.41 ± 6.16 58.16 ± 6.78
# Average droplet diameter (Δdm); ⁎polydispersity index (p¡); zeta potential (ZP); refractive index (RI); viscosity mean (η); SD: standard deviation.
of SNEDDS. The expected shape of formulation F1 could be oblate spheroid due to its lowest viscosity and presence of Tween-80 micelles as reported previously [25,26]. However, the shape of other formulations (F2–F9) could be more asymmetric due to increase in viscosity at 25±1 °C. 3.5. Self-nanoemulsification efficiency of ultra fine super SNEDDS Optimized ultra fine super SNEDDS were further characterized for the self-nanoemulsification efficiency test. It has been reported that when SNEDDS are infinitely diluted with an aqueous medium or GI fluids, there could be every possibility of phase separation which could lead to precipitation of a drug and loss of therapeutic efficacy and bioavailability because they are formed at a particular concentration of oil, surfactant and water [16]. In the present study, distilled water was used as a dispersion medium for self-nanoemulsification efficiency test because it is well reported that there is no significant difference in the SNEDDS prepared using pharmaceutically acceptable surfactants, dispersed in either water or GI fluids [16,20]. Ultra fine super SNEDDS that passed this test in grades A and B were selected for further evaluation, as grades A and B formulations will remain as SNEDDS when dispersed in GI fluids. All other SNEDDS that were falling in grades C, D and E were discarded for further evaluation (Table 1). 3.6. In vitro dissolution/drug release studies In vitro dissolution/drug release studies were performed to compare the release of IND from nine ultra fine super SNEDDS (F1–F9) and marketed IND capsule, all having same amount (25 mg) of drug. The release of IND from formulation F1 was found to be highly significant (p b 0.05) when compared with other SNEDDS [F2–F9] as shown in Fig. 2. The % of IND that was released from F1 after 60 min. of study was found to be 98.4. 93% of the drug release was obtained from F1 in the first 15 min of study itself compared to other formulations. The lowest % of IND release after 60 min of study was observed with formulation F9 (63.3%) that could be due to highest concentration of oil, higher Smix ratio, largest droplet size & PI and highest viscosity. The lowest % of IND release could also be due to the higher conductivity due to formation of swollen micelles in pseudo-ternary system [21]. The results of in vitro drug release studies were found in accordance with the results of characterization studies. Highest drug release in case of F1 was possible due to smallest droplet size, lowest PI, lowest viscosity and eventually higher surface area in case of F1, which permitted rapid drug release. On the other hand, the % release of IND from marketed IND capsule was found to be 92% after 60 min. of study (Fig. 3). SNEDDS formulations showed their drug release profile in two steps. In the first step, the burst release profile was obtained (from 2 to 15 min of study) as shown in Fig. 2. This burst release phase could be due to the presence of surface associated drug [7]. In the final step, the slow sustained release profile was obtained. These results indicated the diffusion
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solubility of IND in optimized SNEDDS F1 was found to be 41.16 ± 3.14 mg/ml. Therefore around 40 mg of IND could be encapsulated in 1 ml formulation of SNEDDS which is much greater than the single oral dose of IND (25 mg). The solubility of IND in optimized SNEDDS was extremely significant as compared to its aqueous solubility (P b 0.001). The enhancement in solubility was 4573 folds in optimized SNEDDS as compared to its aqueous solubility (0.009 mg/ml). This enhancement in solubility of IND in SNEDDS was possible due to the presence of Tween-80 and Transcutol-HP as both are surfactants and potential solubility enhancers. 4. Conclusions Fig. 2. In vitro dissolution (drug release) profile of IND from nine ultra fine super SNEDDS (F1–F9) in distilled water.
controlled dissolution rate of IND from SNEDDS formulations [7]. However, the drug release pattern was found to be one step (immediate release) in the case of marketed IND capsule as shown in Fig. 3. The initial drug release pattern of IND from marketed IND capsule was found to be much slower than the optimized formulation F1 (Fig. 3). It was also observed that drug release was significantly enhanced by optimized SNEDDS F1, as 93% of IND was released in first 15 min of study as compared to 48% from commercial capsules (p b 0.05) [Fig. 3]. Therefore, this enhanced dissolution of IND from optimized SNEDDS could result in higher in vivo absorption and higher in vivo bioavailability. Our results of in vitro dissolution also supported the hypothesis that nanosized droplets of emulsions can enhance the release of poorly soluble drugs [27]. Therefore, the optimized SNEDDS F1, having higher drug release (98.4%), lowest droplet size (8.7 nm), lowest PI value (0.005), lowest viscosity (24.52 cps), stability of SNEDDS and drug and above all, optimum concentration of surfactants (40%) was selected for further evaluation.
3.7. Determination of IND solubility in distilled water and optimized SNEDDS F1 The main obstacle associated in suitable formulation development is the poor solubility of drugs [16]. The aqueous solubility of drugs is the primary factor for dissolution rate and any drug with aqueous solubility of less than 0.1 mg/ml usually present dissolution rate limitation which in turn result in poor in vivo bioavailability [28]. Therefore, improvement in aqueous solubility of poorly soluble drugs is very important to remove obstacles associated in formulation development. The saturation solubility of IND in distilled water at 37 °C was found to be 0.009 ± 0.004 mg/ml. However, the saturation
Fig. 3. Comparative in vitro dissolution (drug release) profile of IND from optimized SNEDDS F1 and marketed IND capsule in distilled water.
Based on lowest droplet size (8.7 nm), lowest PI (0.005), lowest viscosity (24.52 cp), lowest concentration of oil (10% w/w, including 25 mg of IND), optimum surfactant (Tween-80, 20% w/w) and cosurfactant concentration (Transcutol-HP, 20% w/w), water (50% w/w) and SNEDDS formulation F1 has been optimized as an effective formulation. From these results, it was concluded that the developed SNEDDS could be used as the promising vehicle for solubility and dissolution enhancement of IND. Further pharmacodynamics as well as pharmacokinetic studies on animal/human models are required to exploit full potential of SNEDDS for its commercial exploitation. Conflict of interest statement The authors declare no conflict of interest. The authors alone are responsible for content and writing of the paper. Acknowledgement The authors are thankful to Center of Excellence in Biotechnology Research (CEBR) and Department of Pharmaceutics, King Saud University, Riyadh, Saudi Arabia for providing the necessary facilities to carry out these studies. The authors are also thankful to Gattefosse (France) for donating the gift samples of Labrafil and Transcutol-HP. References [1] A.A. Badawi, H.M. El-Laithy, R.K. El-Qidra, H. El-Mofty, M. El-Dally, Archives of Pharmacal Research 31 (2008) 1040–1049. [2] F. Shakeel, W. Ramadan, M.A. Ahmed, Journal of Drug Targeting 17 (2009) 435–441. [3] E.I. Taha, Scientia Pharmaceutica 77 (2009) 443–451. [4] A. Yokotal, M. Taniguchi, A. Tanaka, K. Takeuchi, Inflammopharmacology 13 (2005) 209–216. [5] G.O. Dengiz, F. Odabasoglu, Z. Halici, H. Suleyman, E. Cadirci, Y. Bayir, Archives of Pharmacal Research 30 (2007) 1426–1434. [6] A.A. Date, M.S. Nagarsenker, International Journal of Pharmaceutics 329 (2007) 166–172. [7] A.M. Villar, B.C. Naveros, A.C. Campmany, M.A. Trenchs, C.B. Rocabert, L.H. Bellowa, International Journal of Pharmaceutics 431 (2012) 161–175. [8] N. Thomas, R. Holm, A. Mullertz, T. Rades, Journal of Controlled Release 160 (2012) 25–32. [9] V.N. Kartsev, S.N. Shtykov, I.V. Bogomolova, I.P. Ryzhov, Journal of Molecular Liquids 145 (2009) 173–176. [10] L. Djekic, M. Primorac, J. Jockovic, Journal of Molecular Liquids 160 (2011) 81–87. [11] M. Fanun, Journal of Molecular Liquids 135 (2007) 5–13. [12] F. Shakeel, S. Baboota, A. Ahuja, J. Ali, M. Aqil, S. Shafiq, AAPS PharmSciTech 8 (2007) E104. [13] H.Y. Karasulu, Z.E. Sanal, S. Sozer, T. Guneri, G. Ertan, AAPS PharmSciTech 9 (2008) 342–348. [14] S. Simovic, H. Hui, Y. Song, A.K. Davey, T. Rades, C.A. Prestidge, Journal of Controlled Release 143 (2010) 367–373. [15] N. Barakat, E. Fouad, A. Elmadany, Pharmaceutica Analytica Acta S2 (2011) E002. [16] S. Shafiq, F. Shakeel, S. Talegaonkar, F.J. Ahmad, R.K. Khar, M. Ali, European Journal of Pharmaceutics and Biopharmaceutics 66 (2007) 227–243. [17] F. Shakeel, Pharmaceutical Development and Technology 15 (2010) 131–138. [18] S. Shafiq, F. Shakeel, Pharmazie 64 (2009) 812–817. [19] C.W. Pouton, Advanced Drug Delivery Reviews 25 (1997) 47–58. [20] S.M. Khoo, A.J. Humberstone, C.J.H. Porter, G.A. Edwards, W.N. Charman, International Journal of Pharmaceutics 167 (1998) 155–164. [21] A.B. Mandal, B.U. Nair, D. Ramaswamy, Colloid and Polymer Science 266 (1988) 575–578.
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Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq
Ultra fine super self-nanoemulsifying drug delivery system (SNEDDS) enhanced solubility and dissolution of indomethacin Faiyaz Shakeel a, b,⁎, Nazrul Haq a, b, Mahmoud El-Badry b, Fars K. Alanazi b, c, Ibrahim A. Alsarra a, b a b c
Center of Excellence in Biotechnology Research, King Saud University, Riyadh, Saudi Arabia Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia Kayyali Chair for Pharmaceutical Technology, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
a r t i c l e
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Article history: Received 10 October 2012 Received in revised form 4 January 2013 Accepted 16 January 2013 Available online 30 January 2013 Keywords: Self-nanoemulsifying drug delivery system Indomethacin Dissolution Solubility enhancement
a b s t r a c t The aim of the present investigation was to develop and evaluate ultra fine super self-nanoemulsifying drug delivery system (SNEDDS) of indomethacin (IND) to enhance its solubility as well as in vitro dissolution rate. Different SNEDDS formulations of IND were prepared by aqueous phase titration method. Prepared SNEDDS were subjected to different thermodynamic stability tests. Thermodynamically stable SNEDDS was selected for self-nanoemulsification efficiency test. Selected formulations were characterized in terms of droplet size distribution, viscosity and refractive index. Finally, selected SNEDDS (F1–F9) were subjected to in vitro dissolution/drug release studies. Droplet size and viscosity of formulation F1 was found to be lowest as compared to other formulations. The results of zeta potential indicated the formation of stable SNEDDS. In vitro drug release studies showed 98.4% release of IND from optimized formulation F1. The initial drug release profile of IND from optimized formulation F1 was found to be much faster than marketed IND capsule. The results of solubility studies showed around 4573 fold enhancement in solubility in F1 formulation compared to its aqueous solubility. These results indicated that developed SNEDDS could be successfully used for self-nanoemulsifying drug delivery of IND in order to enhance its solubility as well as in vitro dissolution rate. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Indomethacin (IND) is a potent lipophilic nonsteroidal antiinflammatory drug (NSAID) which has been recommended for the treatment of various kinds of pain associated with arthritis, gout and collagen diseases [1–3]. Currently many therapies like oral, topical and parenteral/injectional are used clinically for the treatment of these diseases. Like other NSAID, IND produces gastric/peptic ulcers upon frequent/chronic administration [4,5]. As per the biopharmaceutical classification (BCS) system, IND possesses poor solubility and high permeability (BCS class II drug). Poor solubility of drugs could result in poor in vivo absorption and weak bioavailability, therefore enhancement in solubility and dissolution rate is important in formulation development. Ultra fine super self-nanoemulsifying drug delivery systems (SNEDDS) are clear, transparent, isotropic system of drug, oil, surfactant and cosurfactant, which forms ultra fine nanoemulsions (usually less than 50 nm in size) upon mild agitation, followed by dilution with an aqueous media or gastrointestinal
⁎ Corresponding author at: Center of Excellence in Biotechnology Research, King Saud University, Riyadh, Saudi Arabia (KSA). Tel.: +966 537507318. E-mail addresses: [email protected], [email protected] (F. Shakeel). 0167-7322/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molliq.2013.01.008
(GI) fluids [6–8]. In the last decade, many molecular liquid based systems like microemulsions, nanoemulsions and SNEDDS etc. have been investigated as novel nanovehicles for many hydrophobic drugs to enhance their thermodynamic stability, solubilization capacity, in vitro dissolution, drug delivery potential, therapeutic efficacy as well as in vivo bioavailability [1–3,6–12]. Some formulation approaches like emulsions, nanoemulsions, nanoparticles and SNEDDS have been investigated for nasal, trasnsdermal, ocular and oral drug delivery of IND in order to enhance its permeation, solubilization and dissolution [1–3,13–15]. Ultra fine super SNEDDS have not been investigated as nanovehicles for oral delivery of IND. When ultra fine super SNEDDS administered orally, they spread rapidly in an aqueous media (GI fluids) providing the hydrophobic drugs in nanometer size range, which in turn enhance the drug dissolution and bioavailability by increasing their aqueous solubility. Therefore, the aim of present study was to develop, characterize and evaluate ultra fine super SNEDDS for oral delivery of IND in order to enhance its solubility as well as dissolution rate which in turn enhance the bioavailability and reduce adverse effects associated with conventional oral therapy of IND. The significant advantage of the present study is that ultra fine super SNEDDS were prepared by spontaneous emulsification (low energy emulsification) method using those materials which are pharmaceutically acceptable and falls under generally regarded as safe category.
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2. Experimental
required amount of Smix was added with drop-wise water addition till an apparent and clear solution was obtained.
2.1. Materials 2.6. Characterization and optimization of SNEDDS IND was obtained as a gift sample from Alfa Aesar, A Johnson Metthey Company (Ward Hill, MA). Labrafil, Lauroglycol-90, Caproyl-90, Labrasol and Transcutol-HP were kindly gifted by Gattefossé (France). HPLC grade ethyl acetate (EA) and Tween-80 were purchased from BDH chemicals (UK). All these materials were of high purity (>99%). All other chemicals used in the study were of analytical reagent (AR) grade. 2.2. Analytical methodology IND in solubility study samples as well as in dissolution samples was analyzed by in-house developed green reversed phase high performance liquid chromatography (RP-HPLC) method in which an environmentally benign green solvent EA (100%) was used as a mobile phase. Green chromatographic identification of IND was performed on Waters HPLC system (Waters, USA) using Lichrosphere 250 × 4.6 mm reverse phase (RP) C8 column (Phenomenex, USA) having a 5 μm packing as a stationary phase. The elution was performed at a flow rate of 1.0 ml/min at 318 nm. Samples (10 μl) were injected using a Waters auto sampler (unpublished data). 2.3. Screening of components for ultra fine super SNEDDS preparation Pharmaceutically acceptable and generally regarded as safe category components were selected for the development of ultra fine super SNEDDS of IND. Selected oils, surfactants and cosurfactants were screened based on solubility of IND in these components. As per our previous report, highest solubility of IND was obtained in Labrafil (as oil phase), Tween-80 (as surfactant) and Transcutol-HP (as cosurfactant) [2]. Therefore, Labrafil, Tween-80, Transcutol-HP and distilled water were selected as oil phase, surfactant, cosurfactant and aqueous phase respectively for the development of optimal ultra fine super SNEDDS of IND. 2.4. Nanophasic map construction and optimization of ultra fine super SNEDDS In order to find out the concentration range of various components for the existence zones of SNEDDS, pseudo-ternary phase diagrams were constructed using aqueous phase titration method [16–18]. Surfactant and co-surfactant (Smix) were premixed in different weight ratios (1:0, 1:1, 1:2, 1:3, 2:1, and 3:1). The concentration of each Smix was 25 g. For each phase diagram, oil phase and specific Smix ratio were mixed thoroughly in different weight ratios ranging from 1:9 to 9:1. Slow titration with aqueous phase was done for each combination of oil and Smix separately [17]. The physical state of prepared SNEDDS was marked on a pseudo-ternary component based phase diagram with one axis representing the aqueous phase, oil phase and the third representing a mixture of surfactant and cosurfactant at fixed weight ratios (Smix ratio). 2.5. Formulation development IND encapsulated nanoemulsions were prepared as similar to our previous research [2]. From the pseudo-ternary phase diagram, nanophasic regions were pointed out and different formulations from this location were chosen for the present study. Considering phase diagrams, maximum nanophasic regions were exposed by 2:1 ratio of Tween-80 and Transcutol-HP in varying proportions. Almost an entire range of nanoemulsions (NE) occurrence in the phase diagram was taken into account and varied oil compositions with minimum surfactant concentration were selected. 25 mg of IND was taken and
2.6.1. Thermodynamic stability test In search of robust formulation and to overcome the problem of metastable formulation, thermodynamic stability tests were performed [16]. Screened formulations were subjected to different thermodynamic stability tests viz. centrifugation, heating & cooling cycle and freeze– pump–thaw cycles as reported in our previous article [16–18]. 2.6.2. Droplet size and zeta potential measurement Mean droplet size and polydispersity index (PI) of screened SNEDDS (F1–F9) were determined using Brookhaven 90 plus particle size analyzer (Brookhaven Instrument Corporation, NY) which was based on light scattering at room temperature (25 °C) at a scattering angle of 90°. The samples of IND-loaded SNEDDS were suitably diluted with distilled water (1:200), sonicated and filtered through 0.45-μm membrane filter. 3 ml of each SNEDDS was taken in an acrylic square. The refractive index (RI) and viscosity of the medium were set at 1.33 and 0.89 cp, respectively. Zeta potential of screened SNEDDS (F1-F9) was determined using Brookhaven 90 plus zeta potential analyzer (Brookhaven Instrument Corporation, NY) using glass cells. 2.6.3. Viscosity and refractive index measurement Viscosity of IND loaded SNEDDS was measured by Brookfield viscometer at 25 ± 1 °C. Refractive index of SNEDDS was determined without dilution using Abbes type refractometer at 25 ± 1 °C. 2.7. Self-nanoemulsification efficiency test Thermodynamically stable SNEDDS were further subjected to self-nanoemulsification efficiency test. The self-nanoemulsification efficiency of IND loaded SNEDDS was assessed using a standard USP XXII dissolution apparatus 2 as reported previously [16]. The self-nanoemulsification performance of each SNEDDS was visually assessed using different grading systems like grades A, B, C, D and E as reported previously [16,19,20]. Formulations those passed self-nanoemulsification efficiency test in grades A and B were selected for further evaluation. 2.8. In vitro dissolution/drug release studies In vitro dissolution/drug release studies were performed in 900 ml of distilled water using USP XXIV method (Dissolution apparatus # 2, at 50 rpm and 37± 0.5 °C). One ml of each SNEDDS (containing 25 mg of IND) was filled in transparent hard gelatin capsules. Samples (3 ml) were withdrawn at regular time intervals (0, 2, 5, 10, 15, 20, 30, 45 and 60 min) and aliquot amount of distilled water was replaced every time. The release of IND from SNEDDS formulation was compared with the marketed capsule formulation. The samples were analyzed for the drug content using in-house developed green RP-HPLC method at the wavelength of 318 nm. 2.9. Determination of IND solubility in distilled water and optimized SNEDDS F1 The saturated solubility of IND in distilled water and optimized SNEDDS F1 was determined by in house developed RP-HPLC method at 318 nm using pure EA as mobile phase (unpublished data). IND in excess amount was added in distilled water and formulation F1 in glass vials (capacity 5 ml) in triplicate. These glass vials were kept in a mechanical Grant shaking water bath OLS 200 (Grant Scientific, UK) at 37 ± 1 °C for 3 days to get equilibrium. After 3 days, samples were filtered by 0.45-μm membrane filter and diluted suitably with
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distilled water. Filtered and diluted samples were injected in HPLC instrument for the quantification of IND by RP-HPLC method at 318 nm. 2.10. Statistical analysis In vitro drug release data of SNEDDS formulations (F1–F9) & marketed capsule and characterization parameters were statistically evaluated by one way analysis of variance (ANOVA) using Dunnett's test to evaluate the significant differences. The statistical software SPSS Version 11 was used to analyze statistical data. 3. Results and discussion 3.1. Screening of components Based on solubility profile of IND in oil, surfactants and cosurfactant; Labrafil, Tween-80 and Transcutol-HP were selected as oil phase, surfactant and cosurfactant respectively for the development of optimal ultra fine super SNEDDS of IND. Distilled water was selected as aqueous phase. The saturation solubilities of IND in Labrafil, Tween-80 and Transcutol-HP were found to be 35.42± 1.12, 72.23 ± 2.91 and 188.97± 3.29 mg/ml, respectively [2]. 3.2. Nanophasic diagram construction and optimization of ultra fine super SNEDDS Pseudo-ternary phase diagrams for ultra fine super SNEDDS were constructed separately for each Smix ratio for the optimization of formulations (Fig. 1). From the phase diagram, it was clearly observed that Smix 1:0 (Fig. 1a) had very low SNEDDS zones. The maximum concentration of oil that was solubilized by this ratio (1:0) was found to be 9% w/w by utilizing very high percentage of Smix (82% w/w). But in the case of Smix 1:1 (Fig. 1b), when the cosurfactant (Transcutol-HP)
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was used along with the surfactant (Tween-80), the SNEDDS zones were found to be increased significantly as compared to 1:0. The maximum concentration of oil that was solubilized by this ratio in the phase diagram was found to be 22% w/w by incorporating Smix around 52% w/w, itself showed the domination of cosurfactant (Transcutol-HP) along with surfactant in nanosizing. The increased SNEDDS zones in 1:1 ratio could be due to the synergistic effects of surfactant and cosurfactant. When the Smix ratio of 2:1 (Fig. 1c) was studied, the SNEDDS zones were increased further as compared to 1:1 ratio. The maximum concentration of oil that was solubilized by 2:1 ratio was found to be 28% w/w by incorporation of Smix around 41% w/w. When the concentration of the surfactant was increased further with respect to the cosurfactant (Smix ratio 3:1, Fig. 1d), the SNEDDS zones were found to be decreased as compared to 2:1 as well as 1:1 ratio. Possibly, this could be a liquid crystalline phase generated by Tween-80 (surfactant) and it was not pacified by the present amount of Tarnscutol-HP (cosurfactant). This liquid crystalline phase could be due to the significant enhancement in the specific conductivity and viscosity of the systems in the phase diagrams [21,22]. There is no need to increase the concentration of the surfactant with respect to the cosurfactant further because the SNEDDS zone started decreasing now (no need to study the Smix ratio of 4:1 or 5:1). When the concentration of the cosurfactant was increased with respect to the surfactant (Smix ratio of 1:2 and 1:3), no SNEDDS zones were observed in the phase diagrams (data not shown). 3.3. Formulation development With regard to nanophasic maps, good SNEDDS zones were exposed by Smix ratio of 1:1, 2:1 and 3:1. Maximum SNEDDS zones were exposed by the Smix ratio of 2:1. SNEDDS formulations with different formulae from these phase diagrams (1:1, 2:1 and 3:1) were precisely selected on the evidence based performance of thermodynamic stability studies.
Fig. 1. Pseudo-ternary phase diagrams showing SNEDDS area for oil phase (Labrafil), surfactant (Tween-80), cosurfactant (Transcutol-HP) at different Smix ratios [Fig. 1a (1:0), b (1:1), c (2:1), d (3:1)] (Arrows showing increasing concentration of particular component).
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Almost the entire range of SNEDDS occurrence in the phase diagrams was covered and different oil compositions with fixed surfactant concentration (40% w/w) were precisely selected. 25 mg of IND was solubilized in required amount of Smix and oil was added with vortexing, finally distilled water was added in drop-wise manner till a clear and transparent dispersion was obtained. The prepared SNEDDS were tightly sealed in glass vials and stored at ambient temperature.
Table 2 Characterization of screened SNEDDS in terms of droplet size, polydispersity, zeta potential, refractive index and viscosity.
3.4. Characterization and optimization of SNEDDS For the development and optimization of robust SNEDDS formulations, the selected SNEDDS were subjected to different thermodynamic tests. These tests were performed qualitatively to remove metastable and thermokinetically stable SNEDDS. Those formulations, which were found to be stable at these tests, were taken for further optimization (Table 1). Table 2 summarized mean droplet size of selected SNEDDS (F1–F9) ranging from 8.7 to 23.8 nm. These results showed the ultra fine droplet size of all formulations (less than 50 nm) which could have great potential for ultra fine super SNEDDS of IND. The largest droplet size appeared in F9 which could be due to the presence of higher concentration of oil (20% w/w) and higher Smix ratio (3:1) (Table 2). When the concentration of Smix was kept constant at 40% w/w and the oil concentration was increased from 10 to 20% w/w, the droplet size was found to be increased. The droplet size of formulation F1 was found to be lowest (8.7 nm) which could be due to the lowest concentration of oil and lower Smix ratio (1:1). Droplet size of all formulations was found to be increased by increasing the Smix ratio. PI value was found to be b0.6 for all SNEDDS, indicating narrow size distribution. Formulation F1 showed least PI value (0.005) suggesting good uniformity in droplet size distribution. Highest PI value was observed with formulation F9 (0.586). Zeta potential (ZP) values of SNEDDS F1–F9 were found in the range of −30.81 to − 27.87 mV. Formulation F1 had a ZP value of − 28.96 mV (Table 2). There were no significant changes observed in ZP values of all SNEDDS [F1–F9] (p ≥ 0.05). It has been reported previously that ZP values in the range of 25–30 mV in either charge characterizes a stable formulation [23,24]. Therefore, results of ZP measurement indicated stabile formation of all SNEDDS (F1–F9) formulations. The negative net charge on ZP for all SNEDDS was possible due to the presence of fatty acids and esters in the Labrafil which was used as the oil phase for development of SNEDDS. The refractive indices (RIs) of SNEDDS (F1–F9) were found in the range of 1.334–1.339 at 25 °C (Table 2). These values of RI were o/w nature of IND loaded SNEDDS. The RI of formulation F1 was found to be 1.334. The viscosity of optimized SNEDDS (F1–F9) ranged from 24.52 to 58.16 cp. The viscosity results could be correlated with the different contribution of Labrafil, Tween-80 and Transcutol-HP utilized in stabilization of optimized formulation compositions. Viscosity of the formulation F1 was found to be lowest (24.52 cp) as compared to other formulations. The viscosity results could also be correlated with shape and geometry
Table 1 Composition of screened SNEDDS that passed thermodynamic stability and selfnanoemulsification efficiency test in grades A & B. Matrices
F1 F2 F3 F4 F5 F6 F7 F8 F9
Formulation composition (% w/w) Oil phase⁎
Smix phase
Water phase
10 15 20 10 15 20 10 15 20
40 40 40 40 40 40 40 40 40
50 45 40 50 45 40 50 45 40
⁎ Oil phase containing 25 mg of indomethacin.
Smix combination
1:1 1:1 1:1 2:1 2:1 2:1 3:1 3:1 3:1
†
Formulation
Δdm#±SD (nm)
p⁎¡
ZP (mV)
RI† ± SD
η ± SD (cps)
F1 F2 F3 F4 F5 F6 F7 F8 F9
8.70 ± 1.41 14.10 ± 1.73 17.40 ± 2.79 14.0 ± 2.15 15.40 ± 2.21 20.90 ± 3.22 15.70 ± 3.11 21.10 ± 3.54 23.80 ± 3.51
0.005 0.357 0.005 0.095 0.201 0.005 0.312 0.566 0.586
−28.96 −27.87 −29.67 −28.15 −30.21 −30.81 −29.80 −30.44 −29.55
1.334 ± 0.004 1.335 ± 0.005 1.336 ± 0.006 1.334 ± 0.005 1.336 ± 0.007 1.337 ± 0.008 1.338 ± 0.008 1.339 ± 0.009 1.339 ± 0.009
24.52 ± 3.41 34.54 ± 4.92 46.33 ± 5.17 27.56 ± 4.14 42.51 ± 5.62 51.61 ± 6.21 29.78 ± 4.58 47.41 ± 6.16 58.16 ± 6.78
# Average droplet diameter (Δdm); ⁎polydispersity index (p¡); zeta potential (ZP); refractive index (RI); viscosity mean (η); SD: standard deviation.
of SNEDDS. The expected shape of formulation F1 could be oblate spheroid due to its lowest viscosity and presence of Tween-80 micelles as reported previously [25,26]. However, the shape of other formulations (F2–F9) could be more asymmetric due to increase in viscosity at 25±1 °C. 3.5. Self-nanoemulsification efficiency of ultra fine super SNEDDS Optimized ultra fine super SNEDDS were further characterized for the self-nanoemulsification efficiency test. It has been reported that when SNEDDS are infinitely diluted with an aqueous medium or GI fluids, there could be every possibility of phase separation which could lead to precipitation of a drug and loss of therapeutic efficacy and bioavailability because they are formed at a particular concentration of oil, surfactant and water [16]. In the present study, distilled water was used as a dispersion medium for self-nanoemulsification efficiency test because it is well reported that there is no significant difference in the SNEDDS prepared using pharmaceutically acceptable surfactants, dispersed in either water or GI fluids [16,20]. Ultra fine super SNEDDS that passed this test in grades A and B were selected for further evaluation, as grades A and B formulations will remain as SNEDDS when dispersed in GI fluids. All other SNEDDS that were falling in grades C, D and E were discarded for further evaluation (Table 1). 3.6. In vitro dissolution/drug release studies In vitro dissolution/drug release studies were performed to compare the release of IND from nine ultra fine super SNEDDS (F1–F9) and marketed IND capsule, all having same amount (25 mg) of drug. The release of IND from formulation F1 was found to be highly significant (p b 0.05) when compared with other SNEDDS [F2–F9] as shown in Fig. 2. The % of IND that was released from F1 after 60 min. of study was found to be 98.4. 93% of the drug release was obtained from F1 in the first 15 min of study itself compared to other formulations. The lowest % of IND release after 60 min of study was observed with formulation F9 (63.3%) that could be due to highest concentration of oil, higher Smix ratio, largest droplet size & PI and highest viscosity. The lowest % of IND release could also be due to the higher conductivity due to formation of swollen micelles in pseudo-ternary system [21]. The results of in vitro drug release studies were found in accordance with the results of characterization studies. Highest drug release in case of F1 was possible due to smallest droplet size, lowest PI, lowest viscosity and eventually higher surface area in case of F1, which permitted rapid drug release. On the other hand, the % release of IND from marketed IND capsule was found to be 92% after 60 min. of study (Fig. 3). SNEDDS formulations showed their drug release profile in two steps. In the first step, the burst release profile was obtained (from 2 to 15 min of study) as shown in Fig. 2. This burst release phase could be due to the presence of surface associated drug [7]. In the final step, the slow sustained release profile was obtained. These results indicated the diffusion
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solubility of IND in optimized SNEDDS F1 was found to be 41.16 ± 3.14 mg/ml. Therefore around 40 mg of IND could be encapsulated in 1 ml formulation of SNEDDS which is much greater than the single oral dose of IND (25 mg). The solubility of IND in optimized SNEDDS was extremely significant as compared to its aqueous solubility (P b 0.001). The enhancement in solubility was 4573 folds in optimized SNEDDS as compared to its aqueous solubility (0.009 mg/ml). This enhancement in solubility of IND in SNEDDS was possible due to the presence of Tween-80 and Transcutol-HP as both are surfactants and potential solubility enhancers. 4. Conclusions Fig. 2. In vitro dissolution (drug release) profile of IND from nine ultra fine super SNEDDS (F1–F9) in distilled water.
controlled dissolution rate of IND from SNEDDS formulations [7]. However, the drug release pattern was found to be one step (immediate release) in the case of marketed IND capsule as shown in Fig. 3. The initial drug release pattern of IND from marketed IND capsule was found to be much slower than the optimized formulation F1 (Fig. 3). It was also observed that drug release was significantly enhanced by optimized SNEDDS F1, as 93% of IND was released in first 15 min of study as compared to 48% from commercial capsules (p b 0.05) [Fig. 3]. Therefore, this enhanced dissolution of IND from optimized SNEDDS could result in higher in vivo absorption and higher in vivo bioavailability. Our results of in vitro dissolution also supported the hypothesis that nanosized droplets of emulsions can enhance the release of poorly soluble drugs [27]. Therefore, the optimized SNEDDS F1, having higher drug release (98.4%), lowest droplet size (8.7 nm), lowest PI value (0.005), lowest viscosity (24.52 cps), stability of SNEDDS and drug and above all, optimum concentration of surfactants (40%) was selected for further evaluation.
3.7. Determination of IND solubility in distilled water and optimized SNEDDS F1 The main obstacle associated in suitable formulation development is the poor solubility of drugs [16]. The aqueous solubility of drugs is the primary factor for dissolution rate and any drug with aqueous solubility of less than 0.1 mg/ml usually present dissolution rate limitation which in turn result in poor in vivo bioavailability [28]. Therefore, improvement in aqueous solubility of poorly soluble drugs is very important to remove obstacles associated in formulation development. The saturation solubility of IND in distilled water at 37 °C was found to be 0.009 ± 0.004 mg/ml. However, the saturation
Fig. 3. Comparative in vitro dissolution (drug release) profile of IND from optimized SNEDDS F1 and marketed IND capsule in distilled water.
Based on lowest droplet size (8.7 nm), lowest PI (0.005), lowest viscosity (24.52 cp), lowest concentration of oil (10% w/w, including 25 mg of IND), optimum surfactant (Tween-80, 20% w/w) and cosurfactant concentration (Transcutol-HP, 20% w/w), water (50% w/w) and SNEDDS formulation F1 has been optimized as an effective formulation. From these results, it was concluded that the developed SNEDDS could be used as the promising vehicle for solubility and dissolution enhancement of IND. Further pharmacodynamics as well as pharmacokinetic studies on animal/human models are required to exploit full potential of SNEDDS for its commercial exploitation. Conflict of interest statement The authors declare no conflict of interest. The authors alone are responsible for content and writing of the paper. Acknowledgement The authors are thankful to Center of Excellence in Biotechnology Research (CEBR) and Department of Pharmaceutics, King Saud University, Riyadh, Saudi Arabia for providing the necessary facilities to carry out these studies. The authors are also thankful to Gattefosse (France) for donating the gift samples of Labrafil and Transcutol-HP. References [1] A.A. Badawi, H.M. El-Laithy, R.K. El-Qidra, H. El-Mofty, M. El-Dally, Archives of Pharmacal Research 31 (2008) 1040–1049. [2] F. Shakeel, W. Ramadan, M.A. Ahmed, Journal of Drug Targeting 17 (2009) 435–441. [3] E.I. Taha, Scientia Pharmaceutica 77 (2009) 443–451. [4] A. Yokotal, M. Taniguchi, A. Tanaka, K. Takeuchi, Inflammopharmacology 13 (2005) 209–216. [5] G.O. Dengiz, F. Odabasoglu, Z. Halici, H. Suleyman, E. Cadirci, Y. Bayir, Archives of Pharmacal Research 30 (2007) 1426–1434. [6] A.A. Date, M.S. Nagarsenker, International Journal of Pharmaceutics 329 (2007) 166–172. [7] A.M. Villar, B.C. Naveros, A.C. Campmany, M.A. Trenchs, C.B. Rocabert, L.H. Bellowa, International Journal of Pharmaceutics 431 (2012) 161–175. [8] N. Thomas, R. Holm, A. Mullertz, T. Rades, Journal of Controlled Release 160 (2012) 25–32. [9] V.N. Kartsev, S.N. Shtykov, I.V. Bogomolova, I.P. Ryzhov, Journal of Molecular Liquids 145 (2009) 173–176. [10] L. Djekic, M. Primorac, J. Jockovic, Journal of Molecular Liquids 160 (2011) 81–87. [11] M. Fanun, Journal of Molecular Liquids 135 (2007) 5–13. [12] F. Shakeel, S. Baboota, A. Ahuja, J. Ali, M. Aqil, S. Shafiq, AAPS PharmSciTech 8 (2007) E104. [13] H.Y. Karasulu, Z.E. Sanal, S. Sozer, T. Guneri, G. Ertan, AAPS PharmSciTech 9 (2008) 342–348. [14] S. Simovic, H. Hui, Y. Song, A.K. Davey, T. Rades, C.A. Prestidge, Journal of Controlled Release 143 (2010) 367–373. [15] N. Barakat, E. Fouad, A. Elmadany, Pharmaceutica Analytica Acta S2 (2011) E002. [16] S. Shafiq, F. Shakeel, S. Talegaonkar, F.J. Ahmad, R.K. Khar, M. Ali, European Journal of Pharmaceutics and Biopharmaceutics 66 (2007) 227–243. [17] F. Shakeel, Pharmaceutical Development and Technology 15 (2010) 131–138. [18] S. Shafiq, F. Shakeel, Pharmazie 64 (2009) 812–817. [19] C.W. Pouton, Advanced Drug Delivery Reviews 25 (1997) 47–58. [20] S.M. Khoo, A.J. Humberstone, C.J.H. Porter, G.A. Edwards, W.N. Charman, International Journal of Pharmaceutics 167 (1998) 155–164. [21] A.B. Mandal, B.U. Nair, D. Ramaswamy, Colloid and Polymer Science 266 (1988) 575–578.
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